EphB-EphrinB Endocytosis Is Repulsive

Abstract

Interactions between Eph receptor tyrosine kinases and ephrins, their membrane-bound ligands, have been implicated in repulsive cues critical to cell migration and neuronal growth cone guidance. Although ephrinA binding to EphA receptors initiates ephrinA cleavage (allowing the interacting cells to disengage), it has been unclear how binding of ephrinBs to EphB receptors is converted into a repulsive signal (see Wilkinson). Zimmer et al. used immunocytochemistry to show that NIH 3T3 cells stably transfected with either ephrinB1 or EphB2 underwent clustering and internalization of the receptor-ligand complex when stimulated by cells expressing the other protein. Similarly, primary neurons cocultured with 3T3 or HeLa cells that expressed labeled EphB2 or ephrinB1 internalized the labeled protein. When one cellular partner expressed a truncated protein lacking cytoplasmic regions, endocytosis of the complex was undirectional (into the cell expressing the full-length protein); EphB2-mediated endocytosis also required tyrosine kinase activity. The authors used time-lapse microscopy of cells expressing fluorescently labeled ephrin, EphB2, or truncated mutants to show that endocytosis regulated cell detachment and repulsion. Marston et al. used time-lapse microscopy of adjacent Swiss 3T3 cells expressing microinjected EphB4 and ephrinB2 and observed membrane ruffling at EphB4-ephrinB2 contact regions followed by cell retraction. EphB4 underwent tyrosine phosphorylation and was endocytosed into cells expressing EphB4, together with ephrinB ligands and fluorescently labeled membrane from ephrinB-expressing cells. The authors used pharmacological analysis together with expression of a dominant-negative mutant of the Rac guanosine triphosphatase to show that Rac-dependent actin polymerization was required for EphB4 endocytosis and cell retraction. Thus, conversion of the EphB-ephrinB interaction into a repulsive signal appears to require endocytosis of the receptor-ligand complex. M. Zimmer, A. Palmer, J. Köhler, R. Klein, EphB-ephrinB bi-directional endocytosis terminates adhesion allowing contact mediated repulsion. Nat. Cell Biol . 5 , 869-878 (2003). [Online Journal] D. J. Marston, S. Dickinson, C. D. Nobes, Rac-dependent trans-endocytosis of ephrinBs regulates Eph-ephrin contact repulsion. Nat. Cell Biol. 5 , 879-888 (2003). [Online Journal] D. G. Wilkinson, How attraction turns to repulsion. Nat. Cell Biol . 5 , 851-853 (2003). [Online Journal]